Analysis of Stored mRNA Degradation in Acceleratedly Aged Seeds of Wheat and Canola in Comparison to Arabidopsis

Seed aging has become a topic of renewed interest but its mechanism remains poorly understood. Our recent analysis of stored mRNA degradation in aged Arabidopsis seeds found that the stored mRNA degradation rates (estimated as the frequency of breakdown per nucleotide per day or β value) were constant over aging time under stable conditions. However, little is known about the generality of this finding to other plant species. We expanded the analysis to aged seeds of wheat (Triticum aestivum) and canola (Brassica napus). It was found that wheat and canola seeds required much longer periods than Arabidopsis seeds to lose seed germination ability completely under the same aging conditions. As what had been observed for Arabidopsis, stored mRNA degradation (∆Ct value in qPCR) in wheat and canola seeds correlated linearly and tightly with seed aging time or mRNA fragment size, while the quality of total RNA showed little change during seed aging. The generated β values reflecting the rate of stored mRNA degradation in wheat or canola seeds were similar for different stored mRNAs assayed and constant over seed aging time. The overall β values for aged seeds of wheat and canola showed non-significant differences from that of Arabidopsis when aged under the same conditions. These results are significant, allowing for better understanding of controlled seed aging for different species at the molecular level and for exploring the potential of stored mRNAs as seed aging biomarkers.

Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins

Mutations in the fragile X mental retardation 1 gene (FMR1) cause the most common inherited human autism spectrum disorder. FMR1 influences messenger RNA (mRNA) translation, but identifying functional targets has been difficult. We analyzed quiescent Drosophila oocytes, which, like neural synapses, depend heavily on translating stored mRNA. Ribosome profiling revealed that FMR1 enhances rather than represses the translation of mRNAs that overlap previously identified FMR1 targets, and acts preferentially on large proteins. Human homologs of at least 20 targets are associated with dominant intellectual disability, and 30 others with recessive neurodevelopmental dysfunction. Stored oocytes lacking FMR1 usually generate embryos with severe neural defects, unlike stored wild-type oocytes, which suggests that translation of multiple large proteins by stored mRNAs is defective in fragile X syndrome and possibly other autism spectrum disorders.

Fragile X mental retardation protein is a size-dependent translational activator

SummaryFMR1 enhances translation of large neural/oocyte proteinsMutations in the highly conserved Fragile X mental retardation gene (Fmr1) cause the most common inherited human intellectual disability/autism spectrum disorder. Fmr1 is also needed for ovarian follicle development, and lesions are the largest genetic cause of premature ovarian failure (POF). FMR1 associates with ribosomes and is thought to repress translation, but identifying functional targets has been difficult. We analyzed FMR1’s role in quiescent Drosophila oocytes stored prior to ovulation, cells that depend entirely on translation of stored mRNA. Ribosome profiling revealed that in quiescent oocytes FMR1 stimulates the translation of large proteins, including at least twelve proteins whose human homologs are associated with dominant intellectual disability disorders, and 25 others associated with neural dysfunction. Knockdown of Fmr1 in unstored oocytes did not affect embryo development, but more than 50% of embryos derived from stored oocytes lacking FMR1 developed severe neural defects. Fmr1’s previously unappreciated role promoting the translation of large proteins from stored mRNAs in oocytes and neurons may underlie POF as well as multiple aspects of neural dysfunction.

1 Microinjection of CPE-Binding Protein Polyadenylated mRNA Increases Developmental Competence of Bovine Oocytes In Vitro

Developmental competence is acquired during oocyte growth and maturation while oocytes undergo both nuclear and cytoplasmic changes. Completion of oocyte maturation and subsequent embryo development relies mostly on maternally synthesised and stored mRNAs at the transcriptionally quiescent phase. The temporal and spatial post-transcriptional and translational regulation of the stored mRNA in mammalian oocyte cytoplasm is essential for developmental competence of oocytes and is often controlled via cytoplasmic polyadenylation. Cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) is required for polyadenylation of most mRNAs during oocyte maturation. It has been reported that in vitro-matured oocytes with high developmental competence showed an increased level of CPEB mRNA in oocyte cytoplasm. Thus, we hypothesise that the introduction of exogenous CPEB mRNA into in vitro-matured oocytes could increase their developmental capability. In this study, we first synthesised polyadenylated CPEB mRNAs by in vitro transcription. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and subjected to in vitro maturation for 21 h. After the removal of cumulus cells, matured oocytes were parthenogenetically activated (5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL−1 cycloheximide). Each activated oocyte was injected with 5 to 10 pL of poly(A)-RNA solution (400 ng μL−1; CPEB mRNA and green fluorescent protein (GFP) mRNA for the injection group or GFP mRNA for the control group) using a micromanipulator. After injection, the oocytes were cultured in SOF medium supplemented with amino acids for 8 days. No difference was observed in cleavage rate between CPEB and control group. However, the blastocyst rate was significantly higher in the CPEB group than in the control (24.9 ± 2.9% v. 15.0 ± 4.5%; P < 0.05). Cleavage and blastocyst rates were analysed by one-way ANOVA. We also compared the gene expression profile of blastocysts derived from both groups. The blastocysts were collected individually and analysed by single-embryo RT-PCR. Twenty-two genes were selected for analysis based on their roles in genomic reprogramming and embryonic development and fell into 6 functional categories: growth regulatory factors, cell cycle regulation, imprinting, apoptosis, pluripotency and DNA methyltransferase. The single-embryo RT-PCR was performed using the Flex-Six integrated fluidic chip (Fluidigm Corp., South San Francsisco, CA, USA) on the BioMark platform (Fluidigm Corp.). Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. We found that 6 genes (H19, GRB10, DNMT1, CCNB1, CDK2, and SOX2) were up-regulated and 3 were down-regulated (DNMT2, BAX, and P53), along with the overexpression of CPEB gene (P < 0.05). Our results demonstrate that developmental competence can be improved by injecting exogenous CPEB mRNA into in vitro-matured metaphase II cattle oocytes, which reaffirms the essential role of CPEB in early embryonic development.

Inflammatory Cytokines and Thrombosis

Abstract SCI-44 Platelets circulate in abundance in a quiescent state, yet are readily activated in physiologic control of blood loss and support for vascular integrity, with excessive or prolonged responsiveness underlying a range of cardiovascular events. Platelet activation is initiated by soluble agonists, including thrombin, platelet-activating factor, and ADP, and is accompanied by adhesion to injured vascular walls or to other circulating inflammatory cells. Activated platelets produce thromboxane A2, ATP and ADP, growth factors, and cytokines such as CD40 ligand, and they shed highly thrombotic microparticles. Anucleate platelets cannot generate new RNA to alter their proteome, but they store factors for prompt release after stimulation. Despite the handicap of lacking a nucleus to direct RNA transcript formation, platelets do, in fact, produce a number of new proteins after appropriate stimulation. The questions, then, are: how do platelets respond to inflammatory signaling in the absence of transcription factors that integrate so much of inflammatory signaling? How are new proteins produced in the absence of stored mRNA? To what events do new platelet proteins contribute? The answers to these questions derive both from stimulated translation of stored mRNA in unactivated platelets and from the presence of unspliced heteronuclear RNA transcripts of select genes, accompanied by stimulated posttranscriptional splicing that removes introns from stored heteronuclear RNA to generate functional mRNA, followed by stimulated translation to produce new protein. These mechanisms are retained in murine platelets, suggesting a continued role for the unique process of posttranscriptional protein production by activated platelets. Thrombin stimulates mTOR and S6 kinase signaling to stimulate spliceosome activity, while platelets respond to lipopolysaccharide through their TLR4 receptor, ultimately to stimulate the AKT and JNK kinases and then spliceosome activation. Human and murine platelets store interleukin-1β heteronuclear RNA, and, upon activation, both produce new pro-IL-1β, but also process it via caspase-1 activity to active cytokine. Activated platelets release functional IL-1β, in association with shed microparticles, to active endothelial cells and naïve platelets themselves. Platelets express signaling type 1 receptors for IL-1, and recombinant IL-1α and IL-1β each activate IL-1β RNA processing and cytokine production. Accordingly, specific blockade of IL-1 signaling with IL-1Ra, in clinical use as Anakinra, suppressed platelet stimulation by soluble IL-1, and suppressed microparticles shed from activated platelets. IL-1 signaling additionally affects platelets' structure and their interaction with immobile surfaces. Remarkably, IL-1Ra also abolished platelet stimulation by lipopolysaccharide, showing that platelets amplify TLR4 signaling by the IL-1 signaling axis. IL-1β accumulates in association with platelets within thrombi formed after FeCl3 damage to murine carotid arteries, not by captured or infiltrating mononuclear cells. The process of posttranscriptional RNA splicing and translation uniquely generates the paradigmatic inflammatory cytokine IL-1β in thrombi, and because this process can be tightly targeted by inhibition of posttranslational splicing, the contribution of platelets to vascular remodeling during and after thrombosis may be specifically addressed. Disclosures: No relevant conflicts of interest to declare.

Prevalence of t(12;21)[ETV6-RUNX1]–positive cells in healthy neonates

t(12;21)(p13;q22)[ETV6-RUNX1] is the most common chromosomal translocation in childhood acute lymphoblastic leukemia, and it can often be backtracked to Guthrie cards supporting prenatal initiation and high levels of circulating t(12;21)-positive cells at birth. To explore the prevalence of ETV6-RUNX1–positive cells in healthy neonates, mononuclear cells from 1417 umbilical cord blood samples were isolated within 24 hours from birth and subsequently screened for ETV6-RUNX1 transcripts using a highly sensitive real-time reverse transcription polymerase chain reaction assay. In first-run polymerase chain reaction, 14 samples were positive at levels below 10−5, of which specific hybridization reflecting the relevant genetic region was positive in 9 cases. Repeated analyses using stored mRNA and flowcytometric sorting of a CD19+, CD8+, and CD19−/CD8− subpopulations from cryopreserved mononuclear cells from the same cord blood samples (mean sorted: 18 × 106 cells) revealed no positive findings, which demonstrates that the level and/or frequency of ETV6-RUNX1–positive cells is markedly lower than suggested in previous studies.

245 EFFECT OF BOVINE OOCYTE MATURATION SYSTEM ON RELATIVE ABUNDANCE OF mRNA

The oocyte cytoplasm contains several transcripts that are important for early pre-implantation embryo development, and alterations on the amount of these stored mRNA can disturb oocyte competence. The aim of this study was to evaluate the relative abundance of specific transcripts in oocytes matured in vivo or in vitro. For in vitro maturation, immature oocytes were obtained by ovum pickup from 4 crossbred cows (group 1: G1) or from ovaries collected at a slaughterhouse (group 2: G2) and matured in TCM-199 containing 10% estrus cow serum and 2 μg FSH for 24 h under 5% CO2 in air at 38.5°C. For in vivo maturation, the same crossbred cows used in G1 received a progesterone intravaginal implant (CIDR®, Eazi-Breed CIDRO, São Paulo, Brazil) and 2 mg of estradiol benzoate (Estrogin®, Farmavet, São Paulo, Brazil) on Day 0. On Day 4, cows were superstimulated with 180 mg FSH (Folltropin®, Bioniche, Canada) injected in 6 decreasing doses every 12 h, and on Day 6, the cows received 0.53 mg of sodium cloprostenol (Ciosin®, Cooper, São Paulo, Brazil). On Day 7, CIDR® was removed and 2.5 mg of gonadorelina (Gestran-Plus®, Tecnopec, São Paulo, Brazil) was injected. Ovum pickup was performed 18 h after gonadorelina injection. Oocytes with expanded cumulus cell were then pooled and used as in vivo-matured oocytes (group 3: G3). Oocytes from all groups were denuded and frozen in liquid nitrogen. Pools of 10 oocytes for each group were subject to RNA extraction and reverse transcription. cDNA was amplified by real-time PCR using the beta-actin gene as the endogenous reference. The transcripts analyzed are encoded by TEA domain 2 (TEAD2), high mobility group N1 (HMGN), zygotic arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor-9 (GDF9), and peroxiredoxin 1 (PRDX1) genes. Results were analyzed by REST software v.2 using the pair-wise, fixed reallocation randomization test. Data from G3 were used as calibrator. There was no difference (P > 0.05) on relative abundance of all transcripts between pools of oocytes matured in vitro or in vivo obtained from the same cows (G1 and G3, respectively). However, the relative abundance of GDF9 (0.22 ± 0.04-fold) was less (P < 0.05), whereas the relative abundance of TEAD2 transcripts (4.27 ± 2.14-fold) was greater (P < 0.05) for in vitro-matured oocytes obtained from slaugterhouse ovaries (G2) when compared with in vivo-matured oocytes (G3). No difference (P > 0.05) on relative abundance was found between G2 and G3 for the other genes. These data suggest that in vitro maturation does not alter the relative abundance of some transcripts stored into oocytes when compared with the ones stored in oocytes obtained from the same donors by means of multiple ovulation. Financial support was provided by CNPq and FAPEMIG.

Mago Nashi Is Essential for Spermatogenesis in Marsilea

Spermatogenesis in Marsilea vestita is a rapid process that is activated by placing dry microspores into water. Nine division cycles produce seven somatic cells and 32 spermatids, where size and position define identity. Spermatids undergo de novo formation of basal bodies in a particle known as a blepharoplast. We are interested in mechanisms responsible for spermatogenous initial formation. Mago nashi (Mv-mago) is a highly conserved gene present as stored mRNA and stored protein in the microspore. Mv-mago protein increases in abundance during development and it localizes at discrete cytoplasmic foci (Mago-dots). RNA interference experiments show that new Mv-mago protein is required for development. With Mv-mago silenced, asymmetric divisions become symmetric, cell fate is disrupted, and development stops. The α-tubulin protein distribution, centrin translation, and Mv-PRP19 mRNA distribution are no longer restricted to the spermatogenous cells. Centrin aggregations, resembling blepharoplasts, occur in jacket cells. Mago-dots are undetectable after the silencing of Mv-mago, Mv-Y14, or Mv-eIF4AIII, three core components of the exon junction complex (EJC), suggesting that Mago-dots are either EJCs in the cytoplasm, or Mv-mago protein aggregations dependent on EJCs. Mv-mago protein and other EJC components apparently function in cell fate determination in developing male gametophytes of M. vestita.